Generation of a high-titer packaging cell line for the ... - Nature

Gene Therapy (2007) 14, 1705–1711 & 2007 Nature Publishing Group All rights reserved 0969-7128/07 $30.00 www.nature.com/gt

ORIGINAL ARTICLE

Generation of a high-titer packaging cell line for the production of retroviral vectors in suspension and serum-free media K Ghani1, S Cottin1, A Kamen2 and M Caruso1 Le Centre de Recherche en Cance´rologie de l’Universite´ Laval, L’Hoˆtel Dieu de Que´bec, Centre Hospitalier Universitaire de Que´bec, Que´bec, Canada and 2Biotechnology Research Institute, National Research Council Canada, Montre´al, Que´bec, Canada 1

Several patients with severe combined immunodeficiency-X1 disease and adenosine deaminase deficiency have been cured by retroviral-mediated gene therapy. Despite the earlier success, the production of retroviral vectors for clinical gene therapy is cumbersome, costly and lacks safety features because of the adherent nature of packaging cells and the necessity to supplement the culture media with bovine serum. The aim of this study was to generate a retrovirus packaging cell line that could be used for the production of large clinical batch vectors. Bicistronic vectors containing an internal ribosomal entry site followed by a selection gene were used to express Moloney murine leukemia gag-pol and amphotropic envelope viral proteins

in HEK293 cells. The candidate clone (293GP-A2) that was selected as the packaging cell line could release recombinant green fluorescent protein retroviruses at 4 107 infectious viral particles per ml. Similar titers were achieved after these cells were adapted to grow in suspension and serum-free media. Furthermore, using the same culture conditions viral titers proved to be stable for a 3-month culture period. The 293GP-A2 packaging cell line has the potential to be cultured in bioreactors, opening the possibility for large-scale use of retroviral vectors in late stage clinical trials. Gene Therapy (2007) 14, 1705–1711; doi:10.1038/ sj.gt.3303039; published online 11 October 2007

Keywords: retroviral vectors; packaging cells; suspension culture; serum-free media

Introduction The retroviral vector derived from Moloney murine leukemia virus (MLV) was the first viral vector developed for gene therapy, and it is still the vector of choice in many clinical trials for the treatment of monogenic disorders. It has been used to successfully treat several patients with severe combined immunodeficiency-X1 disease, and two patients with adenosine deaminase deficiency.1–3 Retroviral vectors have also been proposed to genetically modify T cells to re-target their specificity toward tumor cells.4 Recent results of a melanoma clinical trial show the objective regression of cancer lesions in two patients suggesting the therapeutic potential of genetically engineered lymphocytes.5 The large-scale production of retroviral vectors will be necessary to confirm these results in advanced clinical trials that enroll more melanoma patients, and to extend this strategy to other types of cancer patients. Packaging cell lines currently used for the production of recombinant retroviruses lack some safety features, and scale-up cannot be easily achieved for clinical trials Correspondence: Dr M Caruso, Le Centre de Recherche en Cance´rologie de l’Universite´ Laval, L’Hoˆtel Dieu de Que´bec, Centre Hospitalier Universitaire de Que´bec, 9 rue McMahon, Que´bec, Canada G1R 2J6. E-mail: [email protected] Received 18 December 2006; revised 23 July 2007; accepted 23 July 2007; published online 11 October 2007

with a large number of patients. The first generations of packaging cell lines were derived from mouse 3T3 cells that contained a high level of endogenous retroviral sequences with strong homologies to MLV genome.6–11 Recombination between endogenous viral sequences with transfer vectors and/or packaging plasmids could lead to replication-competent retroviruses (RCR).12,13 The more recent generation of packaging cell lines is derived from human cells like HEK293 (embryonic kidney), TE671 (rhabdomyosarcoma) and HT1080 (fibrosarcoma). Unnecessary viral sequences present in packaging plasmids as well as in transfer vectors have been deleted to improve safety.11,14–18 Another important safety consideration while producing retroviral vectors is the presence of serum from animal origin that supplements the culture medium. The use of serum may introduce biological contaminants like proteins, toxins, viruses and prions. Serum is an expensive raw material, and it increases the complexity, duration and cost of downstream processing (purification, concentration and vector recovery). Furthermore, an immune response directed to fetal calf serum (FCS) components has been observed in patients. This biological phenomenon is suspected to play a role in the disappearance of transduced cells.19 It has been reported that the virus production phase is only feasible without serum for a few days.20–26 However, the complete adaptation of retrovirus producer cell lines to serum-free media (SFM) culture has not yet been achieved.

Retroviral vectors produced in suspension without serum K Ghani et al PACKAGING VECTOR IRES ß-globin Intron pMD2.GPiZeo

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Figure 1 Schematic diagrams of plasmids and retroviral construct. (a) pMD2.GPiZeo and (b) pMD2.AiPuro plasmids contains MLV gag-pol and amphotropic env, respectively. Zeocin and puromycin resistance genes are linked to an EMCV internal ribosomal entry site (IRES) sequence. (c) GFP3 is a retroviral vector derived from MLV that contains the herpes simplex virus thymidine kinase (TK) gene followed by an IRES green fluorescent protein (GFP) sequence. Arrows indicate the start site and the direction of transcription.

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Construction of a high-titer 293SF-derived retroviral packaging cell line Moloney murine leukemia virus gag-pol and amphotropic envelope (env) genes containing the minimum viral sequence (from ATG to stop codon) were cloned in expression vectors under the control of the cytomegalovirus immediate early enhancer/promoter region (CMVi.e.) followed by a b-globin intron. Both plasmids are bicistronic; they contain a viral gene followed by an internal ribosomal entry site (IRES) that allows the expression of a selection marker in a cap-independent manner (Figure 1). The packaging vector that expresses gag-pol was stably transfected into 293SF cells, and 12 zeocinresistant clones were isolated and screened for the presence of reverse transcriptase (RT) in the medium. All clones tested had an RT level ranging from 7000 to 30 000 arbitrary units (Figure 2). The GP21C clone had the highest level of RT; it was comparable to the one found with the FLYRD18 packaging cell line,14 and was 15 times higher than the one obtained with PG13 cells34 (data not shown). Although, a high level of gag-pol expression was achieved with GP21C cells, the cellular growth of this clone and 293SF parental cells was comparable. These data demonstrate that 293SF cells are capable to release efficiently MLV virus cores. In a second step, the env expression vector was introduced by transfection into the GP21C clone. Puromycin-resistant clones were isolated and screened for vector production by transient transfection of the

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The culture of packaging cells is not easily scalable using adherent systems (T-flasks, roller bottles, hollow fibers, microcarriers, Cell Factories and packed beds).15,21,25,27–31 On the contrary, a packaging cell line that could grow in suspension would allow vector production in bioreactors with practically no size limit, and with higher cell densities compared to adherent cells that are limited to growth surface.29,32 Several years ago, the production of retroviral vectors from human lymphoid cells that naturally grow in suspension was reported.24,33 However, the viral productivity was low and cells were cultured with media supplemented with serum. In addition, cytokines that are normally released by lymphoid cell lines would have to be removed from viral preparations to avoid unwanted differentiation of target cells or a possible cytotoxicity. Adaptation to suspension culture of established packaging cell lines is highly challenging; it often produces large clumps that lead to a dramatic reduction in viral titers.29 We have recently reported the adaptation to suspension culture of the 293GPG packaging cell line. Nevertheless, this cell line was cultured in presence of serum and the retroviral vector production was limited to a short period of time due to the toxic nature of vesicular stomatitis virus G protein (VSV-G) envelope used to pseudotype vectors.32 The aim of this study was to generate a packaging cell line that would enable the large-scale production of retroviral vectors for gene therapy clinical trials. We report for the first time the construction of a high-titer packaging cell line that produces vectors in suspension and SFM.

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Figure 2 Reverse transcriptase (RT) activity measured from 12 individual gag-pol expressing clones. Values presented are the mean7standard deviation of an experiment done in triplicate.

green fluorescent protein (GFP)-3 transfer vector (Figure 1). A total of 1 ml of viral supernatant from the best clones could infect between 3.6 and 40.3% HT1080 cells (Figure 3). Viruses released from the 293GP-A2 clone were the most efficient to transduce HT1080 cells. This clone was selected as a candidate packaging cell line to produce MLV core pseudotyped with the amphotropic envelope. A GFP3 transfer vector was stably introduced in the 293GP-A2 clone by infection with VSV-G-pseudotyped

Retroviral vectors produced in suspension without serum K Ghani et al

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Figure 3 Screening of 293GP-A clones. A total of 1 ml supernatant of each transfected clone was harvested and used to infect HT1080 cells. After 2 days, target cells were analyzed for GFP fluorescence by FACS analysis. Values presented are the mean of duplicate experiments of the 10 best clones.

retroviruses to assess its potential as a stable retrovirus producer cell line. Cells were then enriched for GFP fluorescence by cell sorting. A titer of 4 107 infectious viral particles (IVP) per ml was obtained with the viral supernatant harvested from 293GP-A2/GFP cells cultured adherently in presence of serum (data not shown). These results indicate that 293GP-A2/GFP cells release high-titer recombinant retroviruses.

Retroviral vector titers from 293GP-A2/GFP cells cultured in suspension and SFM All the packaging cell line construction steps were performed with cells grown adherently and in presence of serum. We next took on the challenge to produce retroviral vectors from the 293GP-A2 packaging cell line in suspension and SFM. For this purpose, 293GP-A2/ GFP cells had to be adapted to SFM culture. Cells were first cultured adherently with SFM+5% newborn calf serum (NCS). After three passages cells were transferred into a shake flask at 1 106 cells per ml and cultured under conditions described in Materials and methods section. Cell aggregation without drop of cell viability was observed within the first days. After a 2-week culture, the serum concentration was decreased to 1% for 2 weeks until it was completely removed from SFM. Contrary to the suspension adaptation phase, a ‘crisis’ manifested by a drop in cell viability and/or growth rate was observed during the first passages at low-serum concentration. Nevertheless, the culture was able to recover from these crisis events (Figure 4). The complete absence of serum led to smaller size cell aggregates (single cells and/or small aggregates with less than five cells) depending on the cell density with an overall viability above 95%. The adaptation to suspension and SFM was reproduced with a 293GP-A2 packaging cell line expressing Luc3, a bicistronic vector containing a luciferase gene and a Neor gene (data not shown).

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Figure 4 Adaptation of 293GP-A2/GFP cells to growth in suspension and SFM. 293GP-A2/GFP cells were detached from a monolayer culture by trypsinization and resuspended in LC-SFM containing 5% NCS. Cells were continuously maintained in suspension culture and the medium was changed every 3 days. The NCS concentration was reduced to 1% and to 0% during a 1 month period. Arrows indicate the time of cell dilution. (’) Cell concentration, (J) viability.

The ability of 293GP-A2/GFP cells to grow and produce vectors in SFM was investigated. 293GP-A2/ GFP cells were inoculated in shake flasks at a concentration of 1 106 cells per ml and were submitted to a daily medium replacement. Cellular growth of retrovirus producer cells and viral yields were carefully monitored during a 6-day period. The growth of 293GP-A2/GFP cells reached a maximum cell number of 4.7 106 cells per ml at day 6. Viral titers were superior to 107 IVP per ml from day 2 to day 6 with a maximum level of 4 107 IVP per ml at day 6 (Figure 5a). Thus, at similar cellular concentrations, titers obtained in suspension with SFM were identical to those obtained with cells cultured adherently in presence of serum (4 107 IVP per ml). Despite stringent culture conditions (suspension without serum, and without puromycin or zeocin), the retroviral packaging cell line proved to be stable since viral titers were above 107 IVP per ml during a 3-month culture period (Figure 5b). Optimal culture conditions of 293GP-A2/GFP cells were determined by testing the impact of cell density and harvest time on vector production. Viral supernatants were collected at three different cell densities and at 8, 12 and 24 h time points. The maximum viral titer was achieved at 6 106 cells per ml at all time points, with an average value of 3.6 107 IVP per ml. The increase in cell concentration from 1 106 to 3 106 cells per ml led to a parallel threefold increase in viral titer. In contrast, increasing the concentration from 3 106 to 6 106 cells per ml led only to a 1.4-fold increase in viral titer. Similar titers were obtained at the three collection times for each cell density. Titer values at 8 and 12 h were identical to those obtained at 24 h indicating that an 8-h viral harvest would lead to a higher viral yield (Table 1). Although the 293GP-A2 packaging cell line was constructed with plasmids that do not contain overGene Therapy


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Transduction efficiency of retroviral vectors produced from the 293GP-A2/GFP producer cell line The transduction efficacy of retroviral supernatants produced by 293GP-A2/GFP cells grown in suspension with SFM was tested on three adherent and two lymphoid cell lines. The three adherent cell lines were highly transducible: mouse 3T3 cells were transduced at 95.7%, and human HT1080 and TE671 cells were infected at 88.3 and 72.2%, respectively. DG75 (B lymphoid) and Jurkat (T lymphoid) cell lines were, as expected for suspension cells, less infectable with 16.3 and 12.6% transduction efficiency, respectively (Figure 6). These results show that viruses produced in suspension and SFM from 293GP-A2/GFP cells can transduce target cells efficiently.

Discussion Table 1 Influence of harvest time and cell density on vector production Titer (107 IVP per ml)

Harvest time (h)

8 12 24

1 106 cells per ml

3 106 cells per ml

6 106 cells per ml

0.670.02 0.670.02 1.270.03

2.770.05 2.970.12 2.170.05

3.670.03 3.570.10 3.670.07

293GP-A2/GFP cells were inoculated at 6 105 cells per ml, in 20 ml of LC-SFM using 125 ml shake flasks. When the cell density reached 1 106 cells per ml, virus production was performed in three different shake flasks during an 8, 12 and 24 h period of time. The same culture was then used to reach a cell density of 3 106 and 6 106 cells per ml. A similar virus production protocol was also performed at this two-cell concentration. Values presented are the mean7s.d. of three independent experiments.

lapping retroviral sequences, the absence of RCR in 293GP-A2/GFP cells was confirmed using a RCR assay (see Materials and methods section). Gene Therapy

The development of a packaging cell line that grows in suspension and SFM is important for the implementation of late phase clinical trials. In this study, we report the construction of a retroviral packaging cell line derived from 293SF cells. 293GP-A2/GFP cells could produce retroviral vectors at titers up to 4 107 IVP per ml in suspension and SFM. Gag-pol and env plasmids used to generate the 293GP-A2 packaging cell line contained an IRES followed by a selection marker. Bicistronic vectors were used to prevent the loss of viral proteins that were expressed at high levels in 293GP-A2 packaging cells. The retroviral packaging cell line proved to be stable (Figure 5b), which was most likely conferred by the bicistronic vectors used to express viral proteins.35 At similar cellular concentrations, titers obtained in suspension with SFM were identical to those obtained with cells cultured adherently in presence of serum (4 107 IVP per ml). Furthermore, viral titers from 293GP-A2/GFP cells were equivalent or superior to those obtained with other adherent packaging cell lines.14–16 It is also important to mention that the 293GP-A2/GFP retroviral producer cell line is a bulk


population for the GFP3 vector, and cell cloning has the potential to further increase the titer.16 Gag-pol and env expression plasmids used for the generation of 293GP-A2 cells contain the minimum viral sequence to reduce the risk of generating RCR. Using a standard helper assay, no RCR were detected in viral preparations from 293GP-A2/GFP cells. Titers obtained from 293GP-A2/GFP cells cultured in suspension and SFM were in the range of 107 IVP per ml and equivalent after an 8-h, a 12-h and a 24-h production time (Table 1). These data are in agreement with other studies and reflect the half-life at 37 1C of viral particles that range between 4 and 8 h.29,33,36 At low-cell density, titers increased proportionally to cell concentration, although at higher cell concentrations, from 3 106 to 6 106 cells per ml only a 1.4-fold increase in viral titer was observed. These results suggest that culture conditions at a high cell concentration could be limiting in SFM. Optimization procedures will be tested in the future to increase viral productivity. For example, large-scale vector production with 293GP-A2/GFP cells will be optimized in bioreactors with a perfusion system as we have previously reported in short-term cultures of 293GPG cells.32 The continuous feeding of cells and vector harvest should increase cell growth and viral productivity. Many clinical gene therapy studies are showing efficacy,1–3,5 and the use of bioreactors with cells cultured in suspension and SFM could be the ideal setup for large-scale vector production needed for patient treatments. Recombinant retroviruses can incorporate heterologous envelope glycoproteins at their surface. This process known as pseudotyping is used to increase viral infectivity and modify vector tropism. New packaging cell lines could be derived from the GP21C clone to produce viruses pseudotyped with the cat RD114 env or the Gibon ape leukemia env. These vectors produced in suspension and SFM could be useful for ex vivo gene therapy approaches that target hematopoietic cells.37–39 In conclusion, this study shows for the first time the engineering of a packaging cell line that produces hightiter retroviral vectors in suspension and SFM. The 293GP-A2 cell line has the potential for the large-scale biomanufacturing of retroviral vectors, and it should be ideal for the implementation of late phase cancer gene therapy clinical trials with a high number of patients.

Materials and methods Plasmids Plasmids used to generate GP21C and 293GP-A2 clones were derived from the 4.2 kbp pMD2.KG plasmid. This vector contains a CMVi.e. promoter followed by a human b-globin intron and a polylinker. Bicistronic vectors derived from pMD2.KG containing an encephalomyocarditis virus (EMCV) IRES followed by a selection gene were used to express gag-pol and the amphotropic env (4070A) viral proteins. pMD2.iZeor and pMD2.iPuror plasmids were constructed by cloning a 0.9 kbp PstI/ XbaI IRES-Zeor cassette and a 1.2 kbp IRES-Puror cassette in pMD2.KG digested in PstI/NheI. The plasmid pMD2.GPiZeor was constructed by cloning a 5.2 kbp NotI/NsiI gag-pol insert in pMD2.iZeor digested by NotI/PstI. The plasmid pMD2.AiPuror was generated by ligating the 1.9 kbp amphotropic env in pMD2.iPuror opened in NotI/PstI.

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Cell culture HT1080 (ATCC CCL-121), TE671 (ATCC CRL-8805), 3T3 (ATCC CRL-1658), 293SF and derivatives cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma, St Louis, MO, USA). These cell lines were maintained in medium supplemented with 10% FCS (Bio Cell, Drummondville, Canada). 293SF and derivative cells were also cultured with NCS (Bio Cell). Jurkat and DG75 cell lines were cultured with RPMI supplemented with FCS. 293GP-A2/GFP cells were also cultured in suspension and SFM. The SFM was a low-calcium-SFM derived from H-SFM (Invitrogen, Grand Island, NY, USA), and was supplemented with 0.1% (v/v) lipid mixture (Sigma), 0.1% (v/v) bovine serum albumin (98% purity; Sigma) and 0.1% Pluronic F-68 (Sigma).40 Cells were routinely cultured in 125 ml shake flasks (Corning, Acton, MA, USA) at a concentration varying from 3 105 to 1 106 cells per ml in a 20 ml final volume. They were kept in suspension at a stirrer speed of 120 r.p.m., 37 1C, 100% humidity and 5% CO2 (Forma Scientific incubator, Marietta, OH, USA) on a Big Bill orbital shaker (Barnstead/Thermolyne, Dubuque, IA, USA). Transfection Gag-pol clones were generated by transfection using the calcium phosphate procedure. Subconfluent 293SF cells plated in a 10-cm Petri dish were transfected with 10 mg of the pMD2.GPiZeor plasmid. After 24 h of transfection, cells were selected in media supplemented with zeocin (400 mg ml1) for 2 weeks. Zeocin-resistant cells were then subsequently cloned by limiting dilution into 96-well plates and screened using a RT assay. The gag-pol clone with the highest level of RT (GP21C) was transfected by calcium phosphate with 10 mg of the pMD2.AiPuror plasmid, and cells were selected for 2 weeks with 0.2 mg ml1 of puromycin. The screening of 293GP-A isolated clones was performed by transient transfection. Subconfluent clones grown in 60-mm dishes were individually transfected by the calcium phosphate procedure with 5 mg of the GFP3 transfer vector,41 and 5 mg of DNA carrier. After 2 days, 1 ml of supernatant of each transfected clone was harvested and used to infect HT1080 cells. Infection and transduction Virus produced by transient transfection of 293GP-A clones was used to infect 4–5 105 HT1080 cells in the presence of 8 mg ml1 polybrene. Cells were then analyzed for GFP fluorescence 2 days after infection by fluorescence-activated cell sorting (FACS) analysis. The 293GP-A2 clone was selected as the packaging cell line candidate after this screening. 293GP-A2/GFP cells were generated by infection of the 293GP-A2 clone with recombinant GFP retroviruses. GFP viruses were produced by transient transfections and were VSV-G pseudotyped. Infected cells were then sorted by FACS for GFP fluorescence. Virus titers were determined by scoring GFP-positive target cells by FACS analysis. Briefly, TE671 cells were inoculated at a density of 4.5 105 cells per well in sixwell plates and cultured in 2 ml of medium overnight. The medium from each well was replaced with 1 ml of serial dilutions of virus supernatants in a 2 ml final volume containing 8 mg ml1 polybrene. After 48 h, cells Gene Therapy


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were trypsinized and analyzed for GFP fluorescence by FACS. Vector titers were calculated as follows: titer ¼ (F Cinf/V) D, where F is the percentage of GFPpositive cells, determined by flow cytometry; Cinf the total number of target cells at the time of infection; V the viral volume applied and D the virus dilution factor. Infections resulting in 2–20% of GFP-positive cells were considered for titer calculation based on the linear range of the assay. For the transduction assays, adherent cells were plated 1 day prior infection at 105 cells per well in a six-well plate. After 24 h cells were infected with 0.5 ml of virus in 2 ml final volume containing 8 mg ml1 polybrene. Lymphoid cell lines were resuspended at 105 cells per well in a 12-well plate, and incubated with 250 ml of virus in 1 ml final volume containing 8 mg ml1 polybrene. GFP fluorescence was analyzed 2 days after infection.

RT assay The presence of RT in the supernatant of gag-pol clones was measured as follows: 5 ml of supernatant was added to 25 ml of a RT master mix containing 20 mCi ml1 dTT32P, 50 mM Tris-Cl, 75 mM KCl, 2 mM DTT, 1 mM MnCl2, 5 mg ml1 poly(rA)+oligo dT and 0.5% (v/v) NP40. The reactions were incubated 4 h at 37 1C in a 96-well plate, and 6 ml of the total volume was spotted on a DE81 filter paper. The filter was then washed five times with 1 SSC for 5 min and twice with 85% ethanol for 5 min. The radioactivity associated to the filter was then revealed and quantified with a phosphorimager. Replication-competent retrovirus assay The RCR assay consisted of the mobilization of a GFP vector. 3T3 cells containing a GFP vector were plated at a density of 3 105 cells per well in six-well plates. The following day, cells were infected with 2.5 ml of virus produced from 293GP-A2/GFP cells cultured in suspension and SFM (108 IVP). As a positive control, 3T3/GFP cells were also infected with serial dilutions of supernatant produced from 3T3 cells chronically infected by a replication-competent ecotropic MLV. Virus was then allowed to replicate on 3T3/GFP cells for 2 weeks. Supernatants were then harvested and used to infect 3T3 cells. RCR was then evaluated by the presence of GFP cells. GFP cells were observed at up to the 108 dilution of the positive control.

Acknowledgements We are grateful to Pedro Otavio de Campos-Lima for critical reading of the manuscript and to Stephen Goff for the MLV provirus used to generate the 3T3 chronically infected cell line. This study was initiated with a grant from the Canadian Institute of Health Research (CIHR) (IC074582). MC is a Senior Research Scholar of the Fonds de la Recherche en Sante´ du Que´bec (FRSQ).

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